The thin layer chromatographic (TLC) method for determining the radiochemical purity of 99Tcm-sestamibi suggested by the manufacturer of the kit is slow and inconvenient. In this study a more rapid reversed phase chromatography (Sep-Pak) technique is compared with the TLC method for measurement of the radiochemical purity of 99Tcm-sestamibi. The levels of free 99Tcm-pertechnetate in 99Tcm-sestamibi were accurately determined using the Sep-Pak technique. However, due to binding of variable amounts of reduced-hydrolysed unbound 99Tcm to the chromatography cartridge, the Sep-Pak method overestimated the radiochemical purity of the radiopharmaceutical by approximately 3%. In practice, this overestimation is not important and the Sep-Pak technique can be used as a rapid method of determining radiochemical purity of 99Tcm-sestamibi.